Endophytic bacillus subtilis rsn5 in mulberry and application thereof

Abstract

This invention belongs to the field of microbial technology, specifically disclosing an endophytic Bacillus subtilis RSN5 of mulberry and its applications. The endophytic Bacillus subtilis RSN5 of mulberry described in this invention was deposited at the China Center for Type Culture Collection on May 6, 2026, with accession number CCTCC NO: M2026888. This endophytic Bacillus subtilis RSN5 of mulberry was isolated from the mulberry cultivar Sha 2×Lun 109, which is susceptible to Ralstonia solanacearum. It possesses the ability to produce protease, cellulase, and siderophores, promoting mulberry growth and enhancing stress resistance. It also exhibits inhibitory effects against various plant pathogens, showing good inhibitory effects against MSG-1, the pathogen causing bacterial wilt of mulberry. Therefore, it effectively controls bacterial wilt of mulberry, providing a new and important resource for the prevention and control of this disease, and has promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and specifically relates to an endophytic Bacillus subtilis RSN5 from mulberry trees and its applications. Background Technology

[0002] Bacterial wilt is caused by Ralstonia solanacearum complex (Ralstonia solanacearum complex Ralstonia solanacearum Ralstonia solanacearum (RSSC) is a typical bacterial disease affecting plants caused by infection with Ralstonia solanacearum. R. pseudosolanaceae Mulberry leaves and branches will wilt but remain green until the leaves fall off and the entire plant dies. This pathogen can spread through infected seedlings, soil, and water, and is characterized by rapid onset, rapid spread, severe damage, and difficulty in control. Known as "mulberry wilt," it is one of the major mulberry diseases that cause devastating damage to silkworm production. Currently, the control of mulberry bacterial wilt mainly relies on cultivating resistant mulberry varieties in wilt-affected areas. However, existing resistant mulberry varieties generally have certain shortcomings. On the one hand, most resistant varieties have problems such as low leaf yield and insufficient leaf quality; on the other hand, resistant mulberry varieties are mostly propagated asexually, which limits their large-scale promotion in production. Therefore, the selection of environmentally friendly and highly effective microbial agents has become an important means of green control of mulberry bacterial wilt.

[0003] Plant endophytes, first proposed by the German scientist De Barry, refer to a group of microorganisms that live in plant organs, tissues, and intercellular spaces, do not cause disease, and can form a dynamically balanced symbiotic relationship with the host. Due to their safety, efficiency, and availability, plant endophytes have gradually become an important resource for controlling plant diseases. The mechanisms by which plant endophytes control plant diseases mainly include: antagonistic effects, competitive effects, induction of systemic resistance in plants, and promotion of plant growth.

[0004] Bacillus subtilis is an ideal biocontrol bacterium, widely distributed in nature. It possesses advantages such as environmental friendliness, simple culture conditions, broad antibacterial spectrum, and the ability to produce highly resistant spores, making it highly favored by researchers both domestically and internationally. It has been prepared into various biological agents for the biological control of a variety of plant diseases. Currently, wettable powders are the main formulation of microbial agents in my country. Wettable powders are preparations made by mixing microbial raw powder with carriers and adjuvants, followed by pulverization. They can be dispersed in water to form a suspension for spraying. Wettable powders are low in cost, have a high content of active ingredients, and are simple to prepare.

[0005] Although microbial agents and Bacillus subtilis have shown promising prospects in the control of plant diseases, existing microbial agents for mulberry bacterial wilt still have significant technical shortcomings. For example, conventional microbial agents have disadvantages such as weak colonization ability, poor host affinity, and unstable control effect, which cannot meet production needs.

[0006] In summary, existing methods for controlling bacterial wilt of mulberry trees all have shortcomings: the breeding cycle for resistant varieties is long, yield and quality are poor, and their promotion is limited; chemical control does not meet the requirements of green agriculture; and existing microbial agents have problems such as weak colonization ability, poor host affinity, and unstable control effects. Therefore, screening a Bacillus subtilis strain with high affinity for mulberry trees, strong colonization ability, and highly effective inhibitory effect against Ralstonia solanacearum, and developing a targeted and effective microbial agent, is of significant practical importance and application value for solving the problem of bacterial wilt control in mulberry trees and promoting the green and sustainable development of the sericulture industry. Summary of the Invention

[0007] This invention isolates Bacillus subtilis RSN5 from the mulberry cultivar Sha 2×Lun 109, which is mainly promoted for planting but susceptible to bacterial wilt. This strain has the ability to produce protease, cellulase, and siderophores, promotes mulberry growth and enhances stress resistance, inhibits various plant pathogens, and has a good inhibitory effect on MSG-1, the bacterial wilt pathogen of mulberry. It can solve the problem of biological control of bacterial wilt of mulberry and the problem of poor affinity with the main cultivated cultivar, providing an important resource for the application of microbial agents to control bacterial wilt of mulberry in production.

[0008] In view of this, the purpose of the present invention is to provide an endophytic Bacillus subtilis RSN5 of mulberry trees and its application.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a Bacillus subtilis endophytic strain RSN5 from mulberry trees, which was deposited at the China Center for Type Culture Collection on May 6, 2026, with accession number CCTCC NO: M2026888.

[0010] Furthermore, the 16S rDNA sequence of the endophytic Bacillus subtilis RSN5 from the mulberry tree is shown in SEQ ID No. 1. gyrA The sequence is shown in SEQ ID No. 2.

[0011] Of course, the present invention also provides the application of the above-mentioned endophytic Bacillus subtilis RSN5 of mulberry in inhibiting the bacterial wilt of mulberry MSG-1.

[0012] The present invention also provides a biological agent for inhibiting the bacterial wilt of mulberry MSG-1, characterized in that it contains the above-mentioned endophytic Bacillus subtilis RSN5 of mulberry.

[0013] This invention also provides the application of the above-mentioned endophytic Bacillus subtilis RSN5 in mulberry trees and the above-mentioned biological agents in the preparation of products for the prevention and control of bacterial wilt of mulberry trees.

[0014] The present invention also provides a product for preventing and controlling bacterial wilt of mulberry trees, the product comprising the above-mentioned endophytic Bacillus subtilis RSN5 of mulberry trees or the above-mentioned biological agents.

[0015] Furthermore, the product is a wettable powder containing the aforementioned endophytic Bacillus subtilis RSN5 from mulberry trees, as well as a carrier, wetting agent, dispersant, stabilizer, and UV protectant.

[0016] Furthermore, the wetting agent is sodium dodecylbenzenesulfonate, the dispersant is polyvinyl alcohol, the stabilizer is sodium carboxymethyl cellulose, and the ultraviolet protectant is dextrin.

[0017] Furthermore, the wettable powder formulation by weight is as follows: 10% wetting agent, 10% dispersant, 2% stabilizer, 0.1% UV protectant, and the remainder being a carrier and endophytic Bacillus subtilis RSN5 from mulberry trees.

[0018] Furthermore, the wettable powder contains ≥7.9 × 10⁻⁶ spores of Bacillus subtilis RSN5 from mulberry trees. 10 CFU / g, contamination rate ≤2.2%.

[0019] The present invention also provides a method for preventing and controlling bacterial wilt of mulberry trees, the method comprising applying the above-mentioned endophytic Bacillus subtilis RSN5 of mulberry trees or the above-mentioned biological agents or the above-mentioned products to mulberry trees or mulberry rhizosphere soil for the prevention and control of bacterial wilt of mulberry trees.

[0020] The beneficial effects of this invention are as follows: This invention isolates Bacillus subtilis RSN5 from the mulberry cultivar Sha 2×Lun 109, which is mainly promoted for planting in production but is susceptible to bacterial wilt. This strain has the ability to produce protease, cellulase, and siderophores, which can promote the growth of mulberry trees and enhance their resistance to stress. It has inhibitory effects on a variety of plant pathogens and has a good inhibitory effect on MSG-1, the bacterial wilt pathogen of mulberry trees. It can effectively control bacterial wilt of mulberry trees and provides a new and important resource for the prevention and control of bacterial wilt of mulberry trees, with good application prospects. Attached Figure Description

[0021] Figure 1Morphological characteristics of mulberry endophytic bacteria RSN5; where A: colony morphology of mulberry endophytic bacteria RSN5 after incubation on LB solid medium at 37 ℃ for 36 h; B: Gram staining; C: spore staining. Scale bar is 20 µm.

[0022] Figure 2 Phylogenetic analysis of Bacillus subtilis RSN5, an endophytic bacterium from mulberry trees; where A: based on 16S rDNA sequence; B: based on... gyrA Sequence analysis.

[0023] Figure 3 The inhibitory effect of Bacillus subtilis RSN5, an endophytic fungus of mulberry, on 10 plant pathogenic fungi; among them, A: Elm bark beetle SWU10; B: Metarhizium anisopliae SWU42;C: F. oxysporum SWU27; D: F. oxysporum SWU24; E: Colletrichum lagenarium SWU8;F: Verticillium dahlia SWU6; G: Alternaria sp. SWU26; H: Rhizoctonia solani SWU22; I: Colletotrichum gloeosporioides SWU28; J: Cochiobolus sativus SWU25; the experimental conditions were 25 ℃ confrontation culture for 7 days.

[0024] Figure 4 The effect of endophytic Bacillus subtilis RSN5 on the growth of mulberry seedlings was investigated. The control group (CK) was sterile water. The scale bar was 10 cm. The mulberry seedlings in the figure represent the growth of seedlings on day 26 of culture.

[0025] Figure 5 The study aimed to assess the control efficacy of endophytic Bacillus subtilis RSN5 against bacterial wilt in mulberry trees. The control group (CK) consisted of sterile water. The scale bar was 10 cm. The mulberry seedlings in the image were inoculated. R. pseudosolanacearum Growth status on day 22 after MSG-1.

[0026] Figure 6 The study aimed to assess the efficacy of a wettable powder of Bacillus subtilis RSN5, an endophytic bacillus of mulberry, against bacterial wilt in indoor potted plants. A: Sterile water control; B: B. subtilis Treatment with a 100-fold dilution of RSN5 wettable powder; C: B. subtle Treatment with a 300-fold dilution of RSN5 wettable powder; D: B. subtilis Treatment with a 500-fold dilution of RSN5 wettable powder; E: treatment with a 500-fold dilution of 10 billion spores / gram of Bacillus subtilis wettable powder; F: treatment with an 800-fold dilution of 50% carbendazim wettable powder; the image shows mulberry seedling inoculation. R. pseudosolanacearum Growth on day 9 after MSG-1 treatment, scale bar is 10 cm. Detailed Implementation

[0027] It should be understood that the embodiments described are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description of the invention still fall within the scope of protection of the present invention.

[0028] It should be noted that all experimental materials in the embodiments of this invention, unless otherwise specified, are commercially available. Experimental methods in the embodiments of this invention, unless otherwise specified, are generally performed according to conventional experimental methods or methods recommended by the manufacturers of the experimental materials. The mulberry variety involved in this invention is Sha 2 × Lun 109, a cross-pollinated, wind-borne plant bred from first-generation hybrids of the genus *Morus*, cultivated by the Sericulture Research Institute of the Guangdong Academy of Agricultural Sciences, and is a publicly available mulberry variety.

[0029] Example 1: Isolation and Identification of RSN5, an Endophytic Bacterium from Mulberry Trees (1) Sample collection The mulberry tree samples infected with bacterial wilt in this invention were collected in November 2021 from the resource garden of the Guangxi Zhuang Autonomous Region Sericulture Technology Extension Station. The mulberry variety was Sha 2 × Lun 109. The geographical coordinates of the collection site were 22°50'N, 108°13'E.

[0030] (2) Isolation and purification of endophytic bacteria Mulberry root samples were cut into approximately 5 cm segments, rinsed thoroughly with tap water, air-dried, and then completely immersed in 75% alcohol. After removal with tweezers, the alcohol was burned off over an alcohol lamp for surface sterilization. To verify the thoroughness of surface disinfection, the mulberry segments were rolled once on LB solid medium, and the plates were incubated at 28 ℃ for 24 h. No colonies were observed, indicating thorough surface disinfection. The surface-sterilized mulberry segments were then sliced ​​into three layers using a sterile scalpel. The resulting fragments were placed on NA medium, and the plates were incubated at 28 ℃. Observations were made daily, and after new colonies appeared, the culture was purified using the streak plate method.

[0031] (3) Strain screening After purifying single colonies, strains were screened by testing their antagonistic activity against Ralstonia solanacearum MSG-1, and finally a strain with antagonistic activity, RSN5, was obtained.

[0032] (4) Morphological observation and physiological and biochemical identification of endophytic bacteria RSN5 Referring to the *Manual of Systematic Identification of Common Bacteria* and the *Bergey's Manual of Bacterial Identification*, the screened endophytic bacterium RSN5 was streaked onto LB agar and incubated at 37 °C for 36 h. Morphological characteristics such as color, shape, and size of individual colonies were observed. Gram staining was performed on the endophytic bacteria after 12 h of incubation, and spore staining was performed after 24-36 h of incubation. The staining results were observed under a microscope. The results showed that RSN5 colonies were milky white, wrinkled on the surface, and highly adhesive. Figure 1 A), is a Gram-positive rod-shaped bacterium ( Figure 1 B), capable of forming spores ( Figure 1 C).

[0033] Using physiological and biochemical reagent kits, the sucrose utilization, mannitol utilization, and nitrate reduction capabilities of endophytic bacteria were determined. The specific results are shown in Table 1 below.

[0034] Table 1. Physiological and biochemical identification results of endophytic bacteria RSN5 Note: +: positive (growth or response); -: negative (no growth or no response).

[0035] (5) Molecular biological identification of endophytic bacteria RSN5 Pipe 30 µL of PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems, USA) into the bottom of a 1.5 mL centrifuge tube. Use an inoculation loop to pick a fresh single colony of endophytic bacteria RSN5 and add it to the centrifuge tube. Mix well with the DNA extraction buffer and incubate at 100 °C for 10 min. Centrifuge at 12000 r / min for 3 min, then aspirate 20 μL of the supernatant, which is the total bacterial DNA. Using total bacterial DNA as a template, 16S rDNA (sequence shown in SEQ ID NO.1) was amplified using universal bacterial primer 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID No. 3) / 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID No. 4); the DNA gyrase A subunit encoding gene was amplified using primer 42F: 5′-CAGTCAGGAAATGCGTACGTCCTT-3′ (SEQ ID No. 5) / 1066R: 5′-CAAGGTAATGCTCCAGGCATTGCT-3′ (SEQ ID No. 6). gyrA (The sequence is shown in SEQ ID No. 2), the amplification system is shown in Table 2, and the amplification conditions are shown in Table 3.

[0036] Table 2 Amplification System Table 3 Amplification conditions are as follows PCR products were detected by 1.0% agarose gel electrophoresis and sent to Chongqing branch of Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. The obtained gene sequences were aligned using BLAST in the NCBI Genbank database. Sequences with high homology were downloaded and multiple sequence alignments were performed on sequences with high homology using MEGA-X software and Clustal W. The phylogenetic tree was constructed using the Neighbor-Joining method.

[0037] Phylogenetic analysis based on 16S rDNA sequences showed that RSN5 is associated with Bacillus subtilis with accession numbers NR 112116.2 and NR116017.1. Bacillus subtilis Clustered on the same minimum branch ( Figure 2 A). Based on gyrA Phylogenetic analysis of the sequences showed that RSN5 is associated with Bacillus subtilis with accession numbers MK775693.1 and EU138629.1. Bacillus subtilis Clustered on the same minimum branch ( Figure 2 B).

[0038] Therefore, based on the morphological, physiological and biochemical characteristics and phylogenetic analysis results of the strain, RSN5 was identified as Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ), named B. subtilis RSN5.

[0039] Example 2: Biological characteristics of endophytic Bacillus subtilis RSN5 in mulberry trees (1) Detection of growth-promoting and stress-resistance properties of endophytic Bacillus subtilis RSN5 in mulberry trees a. Protease production capacity Will B. subtilis RSN5 was inoculated onto LB solid medium and cultured at 37 °C for 24 h. Fresh single colonies were then picked and cultured overnight in LB liquid medium at 37 °C and 180 r / min to achieve a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL, 2 μL of bacterial culture was spotted onto the protease detection medium. After incubation at 28 ℃ for 3 days, it was found that a clear zone was produced. The ratio of the diameter of the clear zone to the diameter of the bacterial cell was 4.83, indicating that it has the ability to produce protease (Table 4).

[0040] b. Cellulase production capacity Will B. subtilisRSN5 was inoculated onto LB solid medium and cultured at 37 °C for 24 h. Fresh single colonies were then picked and cultured overnight in LB liquid medium at 37 °C and 180 r / min to achieve a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL, 2 μL of bacterial suspension was inoculated onto Congo red agar and incubated at 28 °C for 3 days. The results showed... B. subtilis RSN5 can produce a large clear zone on Congo red medium, with a clear zone diameter to cell diameter ratio of 5.56, indicating that it has the ability to produce cellulase (Table 4).

[0041] c. Phosphorus solubility Will B. subtilis RSN5 was inoculated onto LB solid medium and cultured at 37 °C for 24 h. Fresh single colonies were then picked and cultured overnight in LB liquid medium at 37 °C and 180 r / min to achieve a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL, 2 μL of bacterial culture was inoculated onto PVK medium and incubated at 28 ℃ for 3 days. It was found that no clear zone was formed, indicating... B. subtilis RSN5 does not have phosphorus-soluble ability (Table 4).

[0042] d. Potassium solubility Will B. subtilis RSN5 was inoculated onto LB solid medium and cultured at 37 °C for 24 h. Fresh single colonies were then picked and cultured overnight in LB liquid medium at 37 °C and 180 r / min to achieve a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL, 2 μL of bacterial suspension was inoculated onto potassium-solubilizing medium and incubated at 28 °C for 3 days. It was found that no clear zone was formed, indicating... B. subtilis RSN5 does not have potassium solubilizing ability (Table 4).

[0043] e. Nitrogen fixation capacity Will B. subtilis RSN5 was inoculated onto LB solid medium and cultured at 37 °C for 24 h. Fresh single colonies were then picked and cultured overnight in LB liquid medium at 37 °C and 180 r / min to achieve a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL, 2 μL of bacterial suspension was streaked onto Ashby medium and incubated at 28 °C for 3 days. No growth was observed, indicating... B. subtilis RSN5 does not have nitrogen-fixing ability (Table 4).

[0044] f. Iron-producing capacity Will B. subtilisRSN5 was inoculated onto LB solid medium and cultured at 37 °C for 24 h. Fresh single colonies were then picked and cultured overnight in LB liquid medium at 37 °C and 180 r / min to achieve a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL, 2 μL of bacterial culture was inoculated onto CAS medium and cultured at 28 ℃ for 3 days. It was found that it could produce a clear zone, and the ratio of the diameter of the clear zone to the diameter of the bacterial cell was 4.60, indicating that it could produce siderophores (Table 4).

[0045] Table 4. Growth-promoting and stress-resistance capabilities of endophytic bacteria RSN5 Note: The ratio of the diameter of the transparent zone to the diameter of the bacterial cell indicates the degree of growth promotion and stress resistance of the strain; NA: the strain does not have this ability; the data in the table are mean ± standard error.

[0046] (2) Detection of the antagonistic effect of endophytic Bacillus subtilis RSN5 on plant pathogenic fungi in mulberry trees Flat plate confrontation method testing B. subtilis RSN5's inhibitory ability against plant pathogenic fungi... Elm bark beetle SWU10 Fusarium oxysporum SWU27 、F. oxysporum SWU24 、 Colletrichum lagenarium SWU8 、Verticillium dahlia SWU6 、Alternaria sp. SWU26 、 Rhizoctonia solani SWU22 、Colletotrichum gloeosporioides SWU28 and Cochiobolus agricultural SWU25 was inoculated onto PDA solid medium and incubated at 25 ℃ for 1-3 days. Fresh pathogenic fungal discs with a diameter of 5 mm were then inoculated into the center of a 90 mm diameter PDA solid medium plate, with parallel streaks extending 25 mm to both sides of the pathogenic fungal discs. B. subtilis RSN5 single colonies (pre-inoculated on LB solid medium and incubated at 37 ℃ for 24 h), with pathogen-only mycelial discs as a control, were incubated at 25 ℃ for 7 days. The inhibitory effect of endophytic bacteria on various plant pathogens was observed. The colony diameter of the pathogens in the control group was measured using the cross-cross method, while the diameter of the experimental group was measured perpendicular to the control group. B. subtle The diameter of pathogen colonies in the RSN5 inoculation direction is calculated as follows: inhibition rate = (colon diameter of control group - colony diameter of experimental group) / colony diameter of control group × 100%.

[0047] The results showed that B. subtilisRSN5 showed varying degrees of inhibitory effects against 10 plant pathogens, including those against... Elm bark beetle SWU10 、F. oxysporum SWU24 、Colletrichum lagenarium SWU8 、 Alternaria sp. SWU26 and Cochiobolus sativus The inhibition rate of SWU25 was all above 50%. Figure 3 (Table 5).

[0048] Table 5. Inhibition rate of endophytic Bacillus subtilis RSN5 from mulberry trees against 10 plant pathogenic fungi. Note: The culture was carried out at 25 ℃ for 7 days; the data in the table are mean ± standard error.

[0049] (3) Detection of the antagonistic effect of endophytic Bacillus subtilis RSN5 on Ralstonia solanacearum pathogens R. pseudosolanacearum MSG-1 was streaked onto solid medium B and incubated at 28 °C for 24 h. A single colony was then picked and inoculated into liquid medium B and incubated overnight at 28 °C and 180 r / min to achieve a bacterial suspension concentration of 1 × 10⁻⁶. 8 CFU / mL. Spread 200 μL of bacterial suspension onto B solid medium. After the plate dries, pick up the bacterial suspension with an inoculation loop. B. subtle RSN5 single colonies (pre-inoculated on LB solid medium and incubated at 37 ℃ for 24 h) were spot-cultured on plates and incubated at 28 ℃ for 3 days. Three replicates were set up. The diameter of the inhibition zone and the diameter of the bacterial cell were measured using the cross-cross method. The results showed that... B. subtle RSN5 against pathogens R. pseudosolanacearum MSG-1 exhibits antagonistic activity, with an inhibition zone diameter of 0.17 cm (Table 6).

[0050] Table 6. Plate effect of endophytic Bacillus subtilis RSN5 on Ralstonia solanacearum. Note: Antibacterial effect after 3 days of incubation at 28 ℃; D: diameter of clear zone (cm); d: diameter of bacterial cell (cm); data in the table are mean ± standard error.

[0051] Example 3: Effects of endophytic Bacillus subtilis RSN5 on mulberry seedling growth and disease suppression (1) Effects of endophytic Bacillus subtilis RSN5 on mulberry seedling growth Will B. subtilisRSN5 was inoculated onto LB solid medium and cultured at 37 °C for 24 h. Fresh single colonies were then picked and cultured overnight in LB liquid medium at 37 °C and 180 r / min to achieve a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL. Mulberry seedlings (5-8 true leaves) were treated with 10 mL / plant via root irrigation, with an equal volume of sterile water as a control. Treatment was repeated every 3 days for a total of 3 treatments, with 3 replicates per treatment and 6 seedlings per replicate. Seedlings were cultured in a greenhouse with 14 h light at 30±1 ℃ and 10 h darkness at 25±1 ℃, relative humidity 75%. 26 days after the final endophytic bacterial suspension treatment, the seedlings were removed, the root substrate was carefully shaken off, and the remaining substrate was rinsed with running water. The seedlings were then air-dried, and the plant height, root length, and leaf length and width of the 2nd to 4th leaves (mean values) were measured in different treatment groups. The results showed that the growth of the treated seedlings was significantly better than that of the water control group. Figure 4 As shown in Table 5, B. subtilis The RSN5 treatment group achieved a seedling height of 12.21 cm, which was significantly higher than that of the water control group (10.37 cm) by 17.74%. However, this treatment did not significantly promote the root length, leaf length and leaf width of the seedlings. Although the values ​​of the above indicators were slightly lower than those of the control group, the differences did not reach the statistical significance level (P > 0.05).

[0052] Table 5. Effects of endophytic Bacillus subtilis RSN5 on mulberry seedling growth indicators. Note: CK is the sterile water control group; the data in the table are mean ± standard error, and different letters indicate that the differences between treatments are significant at the 0.05 level.

[0053] (2) The control effect of endophytic Bacillus subtilis RSN5 on bacterial wilt in mulberry trees Will B. subtilis RSN5 was inoculated onto LB solid medium and cultured at 37 °C for 24 h. Fresh single colonies were then picked and cultured overnight in LB liquid medium at 37 °C and 180 r / min to achieve a bacterial suspension concentration of 1×10⁻⁶. 8 CFU / mL. Pathogen R. pseudosolanacearum MSG-1 was streaked onto solid medium B and incubated at 28 °C for 24 h. A single colony was then picked and inoculated into liquid medium B and incubated overnight at 28 °C and 180 r / min to achieve a bacterial suspension concentration of 1 × 10⁻⁶. 8 CFU / mL.

[0054] Will B. subtilisRSN5 was applied to the roots of mulberry seedlings (5-8 true leaves) at a dose of 10 mL per seedling, with an equal volume of sterile water used as a control. Treatment was repeated every 3 days for a total of 3 treatments. The final treatment... B. subtilis After 3 days of root drenching with RSN5 bacterial suspension, inoculate the damaged roots at a rate of 10 mL per plant. R. pseudosolanacearum MSG-1 bacterial suspension. Four replicates were performed for each treatment, with six mulberry seedlings per replicate. Seedlings were cultured in a greenhouse with 14 hours of light at 30±1 ℃ and 10 hours of darkness at 25±1 ℃ and 75% relative humidity. Disease incidence was observed and recorded, with specific disease severity criteria as follows: Grade 0: The plant is healthy and shows no signs of wilting; Grade 1: The plant's leaves begin to wilt and droop, but the leaves are flat, and less than one-third of the leaves have lost water and shriveled. Grade 2: The plant's leaves wilt and droop down to the mulberry stem, with one-third to two-thirds of the leaves losing water and wrinkling. Level 3: More than two-thirds of the plant's leaves have lost water and shriveled; Level 4: The plant is basically withered and dead.

[0055] The disease index, incidence rate, and disease prevention effect are calculated according to formulas (1)-(3).

[0056] A pot experiment on the control efficacy of endophytic Bacillus subtilis RSN5 against bacterial wilt in mulberry seedlings showed that inoculation with the pathogen... R. pseudosolanaceae Following MSG-1, the control group began experiencing symptoms on day 4. B. subtilis The RSN5 treatment group began to show symptoms on day 7. Twenty-two days after inoculation with the pathogen, B. subtilis The incidence rate in the RSN5 treatment group was 33.33%, significantly lower than that in the control group (95.83%) (P < 0.05); the disease index was 31.25, significantly lower than that in the control group (91.67) (P < 0.05). B. subtle The efficacy of the RSN5 treatment group was 65.89% ( Figure 5 (Table 6).

[0057] Table 6. Control efficacy of endophytic Bacillus subtilis RSN5 against bacterial wilt of mulberry. Note: CK is the sterile water control group; the data in the table are mean ± standard error, and different letters indicate that the differences between treatments are significant at the 0.05 level.

[0058] Example 4: Control effect of endophytic Bacillus subtilis RSN5 wettable powder on bacterial wilt in mulberry trees. The following formulation was prepared using silica as the carrier, sodium dodecylbenzenesulfonate (10%) as the wetting agent, polyvinyl alcohol (10%) as the dispersant, sodium carboxymethyl cellulose (2%) as the stabilizer, and dextrin (0.1%) as the UV protectant. B. subtilis RSN5 wettable powder. Its various quality indicators were tested, and the results showed that... B. subtilis The spore content of RSN5 wettable powder is 7.9 × 10⁻⁶. 10 The CFU / g, miscellaneous bacteria rate of 2.2%, pH of 7.7, fineness of 98.2%, drying weight loss of 1.2%, and suspension rate of 93.7% (Table 7) all meet the national standards.

[0059] Table 7 Quality testing of mulberry endophytic Bacillus subtilis RSN5 wettable powder Evaluation under indoor potted conditions B. subtilis The control effect of RSN5 wettable powder on mulberry bacterial wilt was shown in the results, which indicated that root wound inoculation with the pathogen... R. pseudosolanacearum Nine days after the MSG-1 bacterial suspension was applied, all different treatment groups showed control effects against bacterial wilt of mulberry. B. subtilis The incidence rates in the RSN5 wettable powder treatment groups (100x, 300x, and 500x dilutions) were 26.67%, 23.33%, and 50.00%, respectively, all significantly lower than the negative control group (73.33%) (P < 0.05). B. subtilis The incidence rates in the RSN5 wettable powder treatment groups (100 and 300 times dilution) were not significantly different from those in the two positive control groups (26.67% and 33.33%, respectively) treated with 10 billion spores / gram Bacillus subtilis wettable powder at a 500-fold dilution and 50% carbendazim at an 800-fold dilution (P > 0.05). B. subtilis The disease indexes of the treatment groups treated with 100-fold, 300-fold, and 500-fold dilutions of RSN5 wettable powder were 22.50, 18.33, and 35.83, respectively, all significantly lower than those of the negative control group (55.83) (P < 0.05). B. subtilis There was no significant difference in the disease index between the RSN5 wettable powder treatment groups (100 and 300 times dilution) and the positive control group (16.67 times dilution of Bacillus subtilis wettable powder with the lowest disease index) (P > 0.05). B. subtilis The control efficacy against mulberry bacterial wilt was lowest in the 500-fold dilution of RSN5 wettable powder group, at 35.67%, which was significantly lower than that of other treatment groups and the positive control group (P < 0.05). B. subtilisThe control efficacy of RSN5 wettable powder at 100 and 300 times dilution was 60.00% and 67.33%, respectively, which was not significantly different from the positive control group with the highest efficacy at 10 billion spores / gram of Bacillus subtilis wettable powder at 500 times dilution (70.33%) (P > 0.05). Figure 6 (Table 8). The above results indicate that under indoor potted plant conditions, B. subtilis RSN5 wettable powder can effectively control mulberry bacterial wilt, with the best effect when diluted 300 times.

[0060] Table 8. Control efficacy of endophytic Bacillus subtilis RSN5 wettable powder against bacterial wilt in mulberry plants in indoor pots. Note: CK is the sterile water control group; the data in the table are mean ± standard error, and different letters indicate that the differences between treatments are significant at the 0.05 level.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of endophytic Bacillus subtilis RSN5 from mulberry trees, characterized in that, The endophytic Bacillus subtilis RSN5 from mulberry trees was deposited at the China Center for Type Culture Collection on May 6, 2026, with accession number CCTCC NO: M2026888.

2. The application of the endophytic Bacillus subtilis RSN5 of mulberry as described in claim 1 in inhibiting the bacterial wilt of mulberry, MSG-1.

3. A biological agent for inhibiting MSG-1, the bacterial wilt pathogen of mulberry, characterized in that, It includes the mulberry endophytic Bacillus subtilis RSN5 as described in claim 1.

4. The application of the endophytic Bacillus subtilis RSN of mulberry tree as described in claim 1 and the biological agent as described in claim 3 in the preparation of products for the prevention and control of bacterial wilt of mulberry tree.

5. A product for preventing and treating bacterial wilt of mulberry trees, characterized in that, The product includes the endophytic Bacillus subtilis RSN5 of mulberry tree as described in claim 1 or the biological agent as described in claim 3.

6. The product according to claim 5, characterized in that, The product is a wettable powder containing the endophytic Bacillus subtilis RSN5 of mulberry as described in claim 1, as well as a carrier, wetting agent, dispersant, stabilizer and ultraviolet protectant.

7. The product according to claim 6, characterized in that, The carrier is silica, the wetting agent is sodium dodecylbenzenesulfonate, the dispersant is polyvinyl alcohol, the stabilizer is sodium carboxymethyl cellulose, and the ultraviolet protectant is dextrin.

8. The product according to claim 7, characterized in that, The wettable powder formulation, by weight, is as follows: 10% wetting agent, 10% dispersant, 2% stabilizer, 0.1% UV protectant, with the remainder being a carrier and endophytic Bacillus subtilis RSN5 from mulberry trees.

9. The product according to claim 6, characterized in that, In the wettable powder, the spore content of Bacillus subtilis RSN5 endophytic on mulberry trees is ≥7.9 × 10⁻⁶. 10 CFU / g, contamination rate ≤2.2%.

10. A method for preventing and controlling bacterial wilt of mulberry trees, characterized in that, The method includes applying the endophytic Bacillus subtilis RSN5 of mulberry trees as described in claim 1, or the biological agent as described in claim 3, or the product as described in any one of claims 5-9 to mulberry trees or mulberry rhizosphere soil to control bacterial wilt of mulberry trees.

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